Patient Studies

Incidental signal loss in the arterial wall of the aorta and pelvic arteries in patients who had originally received USPIOs (ferumoxtran-10; Sinerem®, Guerbet) for staging lymph node metastases lead to the exciting hypothesis that USPIOs might be used to image macrophage infiltration also in the human vessel wall (Schmitz et al., 2001). Kooi et al. (2003) showed that indeed in patients with severe symptomatic carotid stenosis, ferumoxtran-10 (Sinerem®, Guerbet) uptake predominantly in macrophages of atherosclerotic plaque can be detected as pronounced post-contrast signal loss in gradient echo MR images, even though the dose in patients (2.6 mg Fe/kg) is about 20 times lower than the typical dose used in rabbit studies (Table 5.3). These findings were later confirmed by Trivedi et al. (2004, 2006). Histology showed that USPIO is taken up predominantly by rupture-prone and ruptured lesions (lesions with a thin or ruptured fibrous cap) since 27 of 36 (75 %) of these lesions showed USPIO uptake while this was only the case in 1 of 14 (7 %) of the stable lesions with a thick fibrous cap (Kooi et al., 2003). This indicates that USPIO-enhanced MRI is a highly promising non-invasive method to identify high-risk plaques.

Both Kooi et al. (2003) and Trivedi et al. (2004, 2006) showed that ferumoxtran-10 was primarily taken up by macrophages, confirming earlier findings in animal studies. Double staining for macrophages and iron showed extensive intracellular, granular localization of iron-containing particles in the cytoplasm of macrophages, especially in the vicinity of small capillaries (Figure 5.6) (Kooi et al., 2003). Electron microscopy showed multiple USPIOs per cell in an intracellular granular iron distribution in phagosomes of macrophages (Kooi et al., 2003; Trivedi et al., 2004) (Figure 5.7). Trivedi et al. (2006) reported that focal Perl's staining was usually in the subendothelial fibrous cap region, while diffuse Perl's staining was visualized at every depth in the plaque. The same authors also found that Perl's staining appeared to be intra-cellular as well as extracellular in location (Trivedi et al., 2006). In accordance with earlier animal studies, quite a few histological sections with an abundance of macrophages did not show USPIO uptake, suggesting that functional state of endothelium and macrophages and other factors might determine USPIO uptake (Kooi et al., 2003; Trivedi et al., 2004, 2006). Kooi et al. (2003) also showed

Table 5.3. Summary of human studies investigating uptake of superparamagnetic particles of iron oxide in atherosclerotic plaque.

Type of study iron oxide nanoparticle

Dose

Findings

Reference

Patients who had originally received feramoxtran-10 for lymph node metastases

Symptomatic patients with severe carotid stenosis

Symptomatic patients with severe carotid stenosis

Feramoxtran-10

Feramoxtran-10

Feramoxtran-10

Pronounced signal loss in wall of aortic and pelvic arteries suggests that (Schmitz et al.. feramoxtran-10 accumulates in human atherosclerotic plaques 2001)

Uptake of USPIOs in macrophages of atherosclerotic plaque in 10 of 11 patients (Kooi et al.. Uptake of USPIO in 75 % of the raptured/rapture prone lesions and 7 % of the 2003) stable lesions

Significant signal loss in MR images 24 hours post-infusion but not 72 hours post-infusion

Histology and electron microscopy showed USPIO uptake in macrophages Incidental uptake of USPIO by smooth muscle cells, myofibroblasts, and endothelial cells

Quite a few histological sections with an abundance of macrophages did not show USPIO uptake

Uptake of USPIO in macrophages of atherosclerotic plaque in 7 of 8 patients (Trivedi et al.. Areas of signal reduction in MR images, corresponding to USPIO/macrophage- 2004) positive histological section in all these 7 patients

Histology and electron microscopy showed uptake of USPIOs in macrophages Largest signal loss 24-36 hours post-LTSPIO infusion

Quite a few histological sections with an abundance of macrophages did not show USPIO uptake

Symptomatic patients with severe carotid stenosis

Ferumoxtran-10

Symptomatic patients with severe carotid stenosis

Ferumoxtran-10

Uptake of USPIO in macrophages in 24 of 30 plaques (Trivedi et al..

Areas of signal loss on post-USPIO MR images, corresponding to 2006) USPIO/macrophages-positive histological sections, in 24 of 27 patients Qualitative MRI was highly sensitive (92.5 %) and moderately specific (64 %). Agreement in location of MR signal loss and iron staining was good (Cohen kappa = 0.60)

Significant correlation between magnitude of relative MR signal changes in regions with focal signal loss and iron-positive cell count (;- = —0.63. P < 0.001) Quite a few histological sections with an abundance of macrophages did not show USPIO uptake

19 of 20 patients showed signal loss on the symptomatic as well as on the contralateral asymptomatic side

Figure 5.6. CD68 (a and b), Perl's (c and d), and CD68/Perl's (e) double staining of the endarterectomy specimen of a patient who received USPIOs. Double staining showed colocalization of macrophages and Perl's iron staining. The red coloring (a and b) and brown coloring (e) are indicative of macrophages, and the blue coloring (c, d, and e) is indicative of the accumulation of USPIOs. The double lumen is indicated with L, the surgical cut with C, and tears with T. Reprinted from Kooi et al. (2003), with permission of Lippincott Williams & Wilkins.

Figure 5.6. CD68 (a and b), Perl's (c and d), and CD68/Perl's (e) double staining of the endarterectomy specimen of a patient who received USPIOs. Double staining showed colocalization of macrophages and Perl's iron staining. The red coloring (a and b) and brown coloring (e) are indicative of macrophages, and the blue coloring (c, d, and e) is indicative of the accumulation of USPIOs. The double lumen is indicated with L, the surgical cut with C, and tears with T. Reprinted from Kooi et al. (2003), with permission of Lippincott Williams & Wilkins.

incidental ASMA/Perl's iron and CD31/Perl's iron double staining, which is indicative for limited USPIO uptake by smooth muscle cells, myofibroblasts, and endothelial cells. Trivedi et al. (2006) found no colocalization with smooth muscle and/or endothelial cells, but this can easily be overlooked because the number of these cell types that are Perl's positive is very limited indeed.

A large number of images of atherosclerotic plaque showed pronounced signal loss 24-36 hours post-contrast infusion (Kooi et al., 2003; Trivedi et al., 2006) (Figure 5.8). Since calcified regions in plaque also result in focal signal loss

Figure 5.7. Electron microscopy and Energy-Dispersive X-ray (EDX) analysis of Perl's positive regions of an endarterectomy specimen of a patient who received USPIOs. a Presence of numerous particles within a single macrophage (arrows). The plasma membrane is indicated with PL, the cell nucleus with N, and the extracellular space with ES. b Detail of phagosomes in macrophages containing clusters of iron particles (arrows). c EDX spectrum of particles in macrophages (dotted line). It is shown that these particles contain a large amount of iron. The solid line corresponds to an EDX spectrum of a reference area. Reprinted from Kooi et al.(2003), with permission of Lippincott Williams & Wilkins.

in MR images, only those areas with new focal signal loss in post-contrast images are considered to be a positive MR finding for USPIO uptake. The magnitude of the measured signal loss depends on the used MR pulse sequence as well as whether regions-of-interest contain entire vessel wall or only area of focal or diffuse signal loss. Qualitative MRI was highly sensitive (92.5 %) and moderately specific (64 %) for detection of USPIOs, using histology as standard of reference. The agreement in location of MR signal loss and iron staining was good (Cohen kappa = 0.60) (Trivedi et al., 2006) and there was a significant correlation between the magnitude of relative MR signal changes in regions with

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